SIGNIFICANCE: G protein-coupled receptors (GPCRs) are a large family of membrane proteins that initiate cellular responses to a wide range of extracellular signals, like neutrotransmitters, hormones or photons. Consequently, they are critical for many physiological processes and their dysregulation frequently leads to human disease. This is in good agreement with the fact that >30% of FDA-approved drugs target GPCRs. The main mechanism of action of GPCRs is through the activation of heterotrimeric G proteins, so developing tools to monitor G protein activity with high fidelity and precision is crucial to elucidate the mode of action of many neurotransmitters, hormones or drugs, and to discover novel therapeutic agents. Our goal is to develop a new class of genetically-encoded optical sensors to monitor the activation of heterotrimeric G proteins directly, in real time and at the endogenous level in cells. If successful, our project will deliver high precision G protein activity biosensors that are (1) adaptable to monitor endogenous G protein activity in physiologically relevant experimental systems, (2) readily transferable to other investigators, and (3) scalable for higher throughput. Thus, the biosensors we propose to develop here will have a profound impact in the field by enabling the study of G protein activity under native conditions with unprecedented detail and accuracy. BACKGROUND: The development of fluorescence or bioluminescence resonance energy transfer (FRET or BRET) techniques has been crucial for the advance of the GPCR field by allowing the sensitive and precise measurement of G protein activity in living cells. However, RET-based techniques to measure G protein activation developed to date still have limitations that dampen progress. One is that they rely on the ectopic overexpression of G proteins, thereby compromising the fidelity of readouts. Also, the requirement for simultaneous overexpression of multiple components precludes their use in systems in which genetic manipulation is not easy. Another possible limitation is that they measure G?-G?? subunit dissociation/ rearrangement, a proximal but still indirect measure of G protein activation by GPCRs (which is defined by nucleotide exchange). SYNOPSIS OF AIMS: We have envisioned the design of BRET-based biosensors to monitor endogenous G protein activity by detecting the formation of GTP-bound G? subunits for each one of the 4 G protein subfamilies (Gi/o, Gs, Gq/11, G12/13). Our approach is based on the modular design of biosensors in a two-step process. In the first step (SA#1), we will identify protein modules that specifically bind to active G? subunits of each family and validate that they can report activity when incorporated into a two-component (donor/acceptor) BRET system. In the second step (SA#2), the biosensors will be adapted to a unimolecular system that reports activity by intramolecular BRET in the presence of natively expressed G proteins.